Solid phase extraction method for obtaining high-purity unsaturated compounds or derivatives of said compounds

ABSTRACT

The invention relates to a novel extraction method for obtaining at least one unsaturated, optionally derivatised compound, from mixtures of said compounds with other less saturated constituents, e.g. for obtaining polyunsaturated fatty acids or the derivatives thereof from mixtures with saturated and/or less unsaturated, optionally derivatised fatty acids, by means of selective complexation with a cation exchanger which is partially or fully charged with silver ions, and subsequent decomplexation.

[0001] The present invention relates to a novel extraction method forobtaining one or more unsaturated, derivatized or underivatizedcompounds from mixtures of these with other less highly saturatedcomponents, for example obtaining polyunsaturated fatty acids orderivatives thereof from mixtures with saturated and/or less highlyunsaturated, derivatized or underivatized fatty acids, by selectivecomplexation at a cation exchanger which is partially or completelyloaded with silver ions; and subsequent decomplexation.

[0002] Polyunsaturated long-chain fatty acids (PUFAS) are essentialfatty acids in human metabolism. PUFAs can be subdivided into two largegroups. In addition to the group of ω-6 PUFAs, the formula of which isbased on linoleic acid, there is the group of ω-3 PUFAS, which isbuilt-up, on the basis of α-linolenic acid.

[0003] PUFAs are important building blocks of cell membranes, the retinaand the meninges and are precursors of important hormones, for exampleprostaglandins, thromboxanes and leukotrienes.

[0004] In addition to the function of building blocks, in the course ofrecent years, it has increasingly frequently been found that PUFAsdirectly have a variety of beneficial effects on the human body ordisorders.

[0005] A multiplicity of clinical studies have found that PUFAs, in thecase of, for example, cancer, rheumatoid arthritis, hypertension andneurodermatitis and many other disorders, can make an importantcontribution toward healing or relief. These findings were causallyresponsible for the fact that international institutions and authoritieshave made recommendations controlling the daily intake of PUFAs.

[0006] PUFAs cannot be synthesized by humans de novo, since they lackthe enzyme systems which can introduce a double bond at the >C9 positionin the carbon chain (missing Δ12 desaturase). Humans are only able tosynthesize polyunsaturated fatty acids when there is a supply ofprecursor fatty acids (for example α-linolenic acid) via the diet.However, there is some dispute as to whether this amount is sufficientto cover the requirement of polyunsaturated fatty acids.

[0007] The great majority of essential fatty acids are consumed via thediet. In-particular vegetable oils are enriched with ω-6 fatty acids(for example evening primrose oil contains γ-linolenic acid (GLA)), butonly up to a chain length of C18, and fish oils or oils frommicroorganisms containing ω-3 fatty acids (for example salmon oilcontains eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)). Inprinciple, fish oils and oils from microorganisms are the solecommercial source of polyunsaturated fatty acids. However, generally,the content of the desired PUFA is low and this is present in a mixture,in which case PUFAs which have an antagonistic action can likewise bepresent. In order to consume the recommended daily allowance of PUFAs, alarge amount of oil must be consumed. In particular, this affects thosepatients who have to consume high doses of PUFAs (for example in thecase of cystic fibrosis). To achieve an effect of the individual PUFAswhich is as targeted as possible, enriched or high-purity PUFAs must beused. There is therefore a great requirement for high-purity PUFAs inthe prior art.

[0008] The use of silver-loaded cation exchangers for purifying andseparating unsaturated fatty acids in analytical chromatography systems(column chromatography) has already been described.

[0009] Use is made here not only of HPLC [Adlof, R. O. and E. A. Emken(1985): The isolation of omega-3 polyunsaturated fatty acids and methylesters of fish oil by silver resin chromatography. JAOCS, 62(11),1592-1595; Christie, W. W. (1987): A stable silver loaded column for theseparation of lipids by high performance liquid chromatography. J. ofHigh resolution Chromatography & Chromatography Communications, 10,148-150], SFC [Kadota, Yasuhiko; Tanaka, Isao; Ohtsu, Yutaka; Yamaguchi,Michihiro (1998): Separation of polyunsaturated fatty acids bychromatography using a silver-loaded spherical clay. II Industrial-scalepreparation of high purity DHA. Nihon Yyukagakkaaishi, 47(4), 351-357]and column chromatography systems [Nieto, S.; Ana M Cordoba; J. Sanhuezaand A. Velenzueela (1997): Obtention of highly purified fractions ofeicosapentaenoic acid and docosahexaenoic acid from sardine oil bysilver-resin chromatography: A semi-preparative procedure. Grasas yAceites, 48(4), 197-199; Belarbi, El Hassan; Emilio Molina and YusufChisti (2000): A process for high yield and scalable recovery of highpurity eicosapentaenoic acid esters from microalgae. Enzyme andMicrobial Technology, 26, 516-529] with silica gels but also of ionexchangers. This applies, in particular, to analytical methods.

[0010] A further chromatography method is ligand-exchange chromatography(LEC). This has been used particularly in systems where no PUFAs are tobe separated [Pyell, U., S. Schober and G. Stork (1997): Ligand-exchangechromatographic separation of polycyclic aromatic hydrocarbons andpolycyclic aromatic sulfur heterocycles on a chelating silica gel loadedwith palladium (II) or silver (I) cations. Fresenius J Anal Chem., 359,538-541; Janak, K., M. Demirbueker, I. Haegglund and L. G. Blomberg(1992): Modifications of poly(methyl-3-propylthiol)siloxane to givestationary phases for open tubular supercritical fluid chromatography.Chromatographia, 34(5-8), 335-341].

[0011] Silver-loaded cation exchanger systems are used in theapplication JP 45021376, where Dowex® 50 is described as achromatography material for purifying ethyl ester mixtures based onevening primrose oil. A pure GLAEE fraction (GLAEE=γ-linolenic acidethyl ester) was successfully isolated in 78% yield.

[0012] In all systems the problem occurs that the polyunsaturated fattyacids can be eluted from the column only extremely slowly and poorly(Adlof, R. O.; H. Rakoff and E. A. Emken (1980): Partial ArgentationResin Chromatography (PARC): I. Effect of Percent Silver on Elution andSeparation of Methyl Octadecadienoate Isomers. JAOCS, 9, 273-275). Inthis case, high amounts of solvent are consumed. Furthermore,fractionation steps are required to separate the purified productfractions.

[0013] Only the use of pure acetonitrile (ACN) or mixtures withacetonitrile has led to an acceptable elution time (Dejarlais, W. J.; R.O. Adlof and E. A. Emken (1983): Acetonitrile as Eluent in Silver ResinColumn Chromatography. JAOCS, 60(5), 975-978). Here, generally gradientsystems are used which make regenerating the solvents more difficult.

[0014] For industrial use, large amounts of acetonitrile, owing to thehigh cost and the toxicological risk (acetonitrile can contain prussicacid (HCN)), are highly unsuitable.

[0015] Scaling up the chromatographic methods, and thus industrial use,is also only possible with limitations, owing to the column dimensionsto be used.

[0016] Adlof et al (1980, above) have produced partially silvered cationexchangers which made it possible to elute esters of diunsaturated fattyacids using alcohols as eluents. More highly unsaturated fatty acidshave not been used. However, it must be assumed that likewise in thiscase, without acetonitrile, elution is not possible.

[0017] It is reported that, starting from fish oils for production bymeans of SFC, costs of $550/kg of DHAEE arise [Alkio, M.; C. Gonzalez;M. Jantti and O. Altonen (2000): Purification of polyunsaturated fattyacid esters from Tuna oil with supercritical fluid chromatography.JAOCS, 77(3), 315-321]. Here, a production rate of approximately 0.5 gof DHAEE per kg of stationary phase and hour is mentioned. This isequivalent to a loading (DHAEE/stationary phase) of 0.05%. The capitalcosts of the SFC equipment are $2 million.

[0018] An SFC production method is likewise known using a silver-loadedmatrix starting from fish oil, in which DHAEE can be produced for $4000/kg [Tanaka, Isao (1996): Supercritical chromatography facilitatesfatty acid production. Chem. Eng. April, 19-21].

[0019] Yamamura et al (Yamamura, R. and Y. Shimomura (1997): Industrialhigh-performance liquid chromatography purification of docosahexaenoicacid ethyl ester and docosapentaenoic acid ethyl ester from single celloil. JAOCS, 74(11), 1435-1440) report an HPLC production method startingfrom a single-cell oil in which the production costs were not reported.The DHAEE productivity was 0.1 kg/h. When the column used is considered(approximately 160 kg of silica gel), this gives a productivity of0.0006 kg per kg of stationary phase per hour. This corresponds to aloading of 0.06%. The solvent used was toxic methanol.

[0020] It becomes clear that chromatographic systems encompass amultiplicity of problems for purifying PUFAs and are thereforeunsuitable for producing polyunsaturated fatty acids on a relativelylarge scale (Zhou, Dequan and Xuebing Xu (2000): Enzymatic enrichment ofLong-chain Polyunsaturated Fatty Acids from Fish Oils. Shipin Kexue(Bejing), 21(12), 188-194. Teramoto, M.; H. Matsuyama; N. Ohnishi; S.Uwagawa and K. Nakai (1994): Extraction of ethyl and methyl esters ofpolyunsaturated fatty acids with aqueous silver nitrate solutions. Ind.Eng. Chem. Res., 33, 341-345).

[0021] On the other hand, mild extraction methods could offerconsiderable advantages for obtaining PUFAs from mixtures with othersubstances.

[0022] First approaches toward such aqueous extraction systemscontaining silver nitrate have been investigated (Suzuki, T.; S.Kikuchi; K. Nakano; S. Kato and K. Nagahama (1993): Supercritical fluidextraction of polyunsaturated fatty acid ethyl esters from aqueoussilver nitrate solution. Bioseparation, 3, 197-204, Teramoto et al1994). Kato et al (S. Kato,. N. Kunio, N. Hidetomi and N. Kaoru (1994):Separation and recovery of polyunsaturated fatty acids by emulsion filmmethod. JP06279781) studied the use of oil-in-water emulsions, onlyseparation of the overall PUFA fraction being reported. In this case,however, the use of silver nitrate, which is hazardous to health, is ahindrance for an industrial application. Furthermore, in an aqueousenvironment, a risk of oxidation of the PUFAs is particularly high,which can be further accelerated, in particular, by the presence ofsilver nitrate. The handling of silver nitrate additionally proves to bevery difficult, since silver nitrate can be relatively rapidly oxidizedto silver oxide and is generally considered unstable.

[0023] Solid-phase extraction systems without silver which have alreadybeen described do not show sufficient selectivity in the separation ofPUFAs (Wilson, R.; J. R. Henderson; I. Burkow and J. R. Sargent (1993):The enrichment of n-3 polyunsaturated fatty acids, using aminopropylsolid phase extraction columns. Lipids, 28(1), 51-54). Solid-phaseextraction processes using silver for separating PUFAs, in particularwithout using acetonitrile, are not known to the applicants.

[0024] In addition, purification by facilitated diffusions throughsilver-loaded membranes has been attempted [Shibaki, A.; Y. Irimoto; K.Saito; K. Sugita; T. Baba; I. Honjyo;, S. Moriyama and T. Sugo (1999):Selective Binding of Docosahexaenoic acid ethyl ester to a silverion-loaded porous hollow-fiber membrane. JAOCS, 76(7), 771-775].

[0025] Owing to directive 88/344/EEC of Jun. 13, 1988 on theapproximation of the laws of the Member States on extraction solvents,which must be heeded in the production of foodstuffs and foodingredients, when DHA is produced for the food sector, the use ofacetonitrile must be absolutely excluded.

[0026] In view of said prior art, the object underlying the presentinvention was therefore to provide a novel method for obtainingunsaturated compounds from mixtures with other substances, for exampleother organic saturated or less highly unsaturated compounds.

[0027] This method should make possible a quantitatively sufficientpurification which is better than is described in the prior art, andpermit a simplification of the method and economical design of the same.Moreover, the method should be selective in order to permit, forexample, the separation from one another of unsaturated compounds whichdiffer only slightly structurally.

[0028] A further object of the present invention was to provide a novelmethod for obtaining unsaturated fatty acids or derivatives thereof frommixtures with other substances.

[0029] This method should make possible a quantitatively sufficientpurification of PUFAs which is better than is described in the priorart, and permit a simplification of the method and economical design ofthe same. Moreover, the method should be selective in order, forexample, to make it possible to separate unsaturated fatty acids orderivatives thereof differing structurally only slightly, for exampleDHAEE/DPAEE.

[0030] Preferably, the inventive method is to be suitable for use in thefood sector, and therefore, for example, make it possible to purifyPUFAs from mixtures without the use of acetonitrile or other toxicsolvents as extraction media.

[0031] This object, and also other objects which are not mentionedexplicitly but which can be derived as such or can be inferred from thecontexts discussed at the outset herein, is achieved by a method asclaimed in patent claim 1. Expedient modifications of the inventivemethod are claimed in the subclaims which are referred back to claim 1.

[0032] This is because the inventive object can be achievedastonishingly simply by

[0033] (i) loading a strongly acidic cation exchanger with silver ions,

[0034] (ii) mixing the loaded cation exchanger with a liquid mixturecontaining the unsaturated compound to be purified with or without atleast one solvent,

[0035] (iii) in a batch method, contacting the mixture with theexchanger for a certain time at a defined temperature which is below theboiling point of the solvent used,

[0036] (v) separating off the supernatant, and

[0037] (vi) detaching the unsaturated compound from the ion exchanger.

[0038] In contrast to the abovementioned methods, in the presentapplication, in addition to the silver ions required for the separation,a matrix is used (ion exchanger) which makes a considerable contributionto the selectivity, reaction time and quality of the separation. Thus,in the methods described in the prior art, it can also be observed that,although silver ions are present, no separation of the PUFAs isachieved.

[0039] Depending on the number of double bonds and the field ofapplication (food or drugs) and the solvents thereby approved, differentprocedures are followed in the present method. Thus, fatty acids whichhave fewer than 4 double bonds can in principle be detachedquantitatively from the exchanger without acetonitrile. Fatty acidshaving more than 4 double bonds can only be quantitatively detached fromthe exchanger when acetonitrile (FIG. 1: variant A), the novelregeneration method (FIG. 1: variant B) or a partially silveredexchanger (FIG. 1: variant C) is used.

[0040] Generally, it can be observed that polyunsaturated fatty acidsbind to the exchanger with different strengths. Thus, in some cases,quantitative detachment is observed even in a polar solvent with orwithout additional heating. This can be explained by the differingdegree of complexation of the PUFA. Thus a triunsaturated PUFA can enterinto a maximum of 6 complex bonds and a hexaunsaturated PUFA can enterinto a maximum of 12 complex bonds, and thus form the stronger bondingspecies.

[0041] The unsaturated compound can be detached according to theinvention from the cation exchanger at a temperature of from −20° C. to80° C. Preferably, the unsaturated compound is detached at a temperatureof from −20° C. to 40° C., very particularly preferably at from −20° C.to 10° C. The detachment can be performed according to the invention byadding a solvent selected from the group consisting of ketones(preferably acetone, 2-methyl ethyl ketone), medium-chain alcohols(preferably 2-propanol, propanol or butanol), short-chain alcohols(preferably methanol or ethanol) and nitriles (preferably acetonitrile).

[0042] Compounds which can be isolated according to the invention frommixtures comprise, inter alia, fatty acids (saturated, unsaturated orpolyunsaturated), isomeric fatty acids (cis, trans, conjugated,isolated), fatty acid derivatives (e.g. ethers or esters, for examplemethyl ester, ethyl ester, wax esters, triglycerides, diglycerides,monoglycerides), modified fatty acids (for example hydroxy fatty acids,oxo fatty acids (keto ), amino fatty acids) also as derivatives,unsaturated alkenes, unsaturated alcohols, unsaturated ethers,unsaturated ketones or aldehydes, aromatics, steroids or steroid esters,sugars (monomers, oligomers, . . . ), tocopherols, astaxanthin.

[0043] Fatty acids which can be isolated according to the invention frommixtures with other organic compounds comprise, inter alia:hexadecadienoic acids, hexadecatrienoic acids, hexadecatetraenoic acids,linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid,arachidonic acid, eicosatrienoic acids, eicosatetraenoic acids,eicosapentaenoic acids, docosapentaenoic acids, docosahexaenoic acid,tetracosadienoic acids, octacosaoctaenoic acids and their salts oresters, ethers or other derivatives of these fatty acids.

[0044] Preferably, the fatty acid ethyl esters are purified by thismethod.

[0045] Generally, fatty acids which can be purified according to theinvention are all fatty acids of the chain length C14-C30, orderivatives of such fatty acids, which have more than two double bonds.

[0046] Separation of compounds depending on the position of the doublebonds or on the configuration of the double bonds is possible using theinventive method.

[0047] In principle, when fully silvered exchangers are used, the bestselectivities and the highest loadings of the exchangers with PUFA areachieved.

[0048] According to the invention, the starting mixtures which can beused are natural oils, for example fish oils, vegetable oils,microbially produced oils, processed oils, ester mixtures, free fattyacid mixtures, unsaturated compounds and/or derivatives of suchcompounds.

[0049] Fish oils contain, for example, 10-20% DHA, and microbiallyproduced oils contain up to 60% DHA. Therefore, the oils also containdiffering amounts of fatty acids to be separated off. For a personskilled in the art, this gives the difficulty [lacuna] a specific purityof the pure DHA which is achievable by the purification step. Generally,therefore, it makes more sense to define a purification factor to assessthe quality of the purification. The purification factor, in thesimplest case, can be the quotient of the purity of the product and thepurity of the substrate. The purities can be determined, for example,from gas-chromatographic analysis (unit: area %).

P _(F) =P _(fatty acid in the product) /P_(fatty acid in the substrate)*100%,

[0050] where

[0051] P_(F)=Purification factor (%)

[0052] P=Purity

[0053] The purification factors after a successful purification arealways >1. However, depending on the starting mixture, even highervalues can be achieved; in the case of a starting mixture which has thetarget fatty acid at 0.5% and, after purification, a purity of 95%, forexample 190.

[0054] In the example of DHAEE, a high-purity 95% DHAEE can be producednot only from a 47% pure DHAEE mixture, but also from an 80% pure DHAEEmixture. The purification factors in these examples are between 1.19 and2.02.

[0055] To carry out the inventive method, no chromatography column isrequired and no fractionation in the actual sense needs to be carriedout.

[0056] Before the separation of supernatant and product phase, only asingle equilibrium needs to be established. This is in complete contrastto chromatography (for example HPLC, SFC), in which a separation is onlyachieved after achieving a certain number of plates (for examplecolumn), or after establishing a very high number, in somecircumstances, of equilibria.

[0057] Technical problems which usually occur owing to change of solventin chromatography (air bubbles, swelling of the exchanger,inhomogeneities) do not occur in the inventive method. Likewise, flowproblems (inhomogeneous flow) over the separation column (gradientformation) do not occur. At the same time, higher product purities canbe achieved. It is also possible to set the product purity in a targetedmanner.

[0058] Surprisingly, it has been shown by the abovementioned inventorsthat polyunsaturated fatty acids or derivatives of these polyunsaturatedfatty acids (for example DHAEE) can be complexed in one step from asolution (e.g. DHAEE/DPAEE) in a batch process in a highly selectivemanner on a silver-loaded cation exchanger.

[0059] The selectivity of the complexation may surprisingly becontrolled very simply, depending on the fatty acid concentration to beseparated, the type of ion exchanger used and its amount, the solventused, the temperature, the time and the silver loading of the ionexchanger.

[0060] These relationships can be used in a targeted manner for thepurification. By establishing certain conditions, high-purity PUFAs canbe produced without complex chromatography steps.

[0061] The novel method is therefore not a chromatography method, but aselective extraction method (complexing more highly unsaturated fattyacids/derivatives on the cation exchanger or cation exchangers and asubsequent decomplexation step in a simple stirred vessel in a batchmethod).

[0062] Cation exchangers which can-be used according to the inventionmay be found in the following list, with the usual trade. names beingcited: Dowex® 50 WX8, Dowex® 50 WX4, Dowex® 50 WX2, Dowex® MWC1, Dowex®MSC1, Dowex® Monosphere C-350, Dowex® CCR-2, Dowex® DR 2030, Amberlite®CG50, Amberlite® IR-120, Amberlyst® 15, Bio-Rex® 70 Resin, Macherey &Nagel PS-DVB®. In particular, those cation exchangers which have thefollowing properties can be used:

[0063] Strongly acidic cation exchangers: gels which have as parentsubstance styrene with divinylbenzene branches, have sulfonic acidand/or carboxyl groups as active silver-bearing group and aremicroporous, or are preferably macroporous. In particular,macroreticular ion exchangers are also particularly suitable, since theyare solvent-stable and have a considerably larger surface area comparedwith gels. These likewise bear sulfonic acid and/or carboxyl groups asfunctional groups.

[0064] Amberlyst® 15 and Dowex® DR2030 can be used with particularpreference.

[0065] The loading capacity of the cation exchangers usable according tothe invention ranges in this case from 0.1 to 15% by weight (g ofPUFA/100 g of H⁺ exchanger) of total PUFA.

[0066] This novel method represents, in a surprisingly simple manner, agreat simplification and economical enhancement of the purificationprocesses described to date in the prior art. Also, it is possiblewithout any problems to adapt this method to large-scale and industrialpurposes, since, for example, columns etc. are not used.

[0067] Also, it was completely surprising to observe that the purity ofthe product on the cation exchanger can be markedly increased andselectively controlled by a subsequent washing operation with a solventand subsequent heating in one or more solvents.

[0068] The heat supply can be provided, according to the invention, forexample, by using external heat sources, by electromagnetic radiation,for example microwaves or infrared radiation, or else via ultrasoundtreatment. Any heat sources that are beneficial to the process can beused.

[0069] It is suspected here that, owing to the effect of heat (energy),incompletely complexed fatty acid in which not all double bondingelectrons are present in complexed form (this can be the product fattyacid, but also secondary fatty acids or derivatives) is decomplexed,preferably the more highly unsaturated fatty acid, after decomplexing,being recomplexed to the cation exchanger and occupying the positionswhich have become free there. The secondary fatty acid, which islikewise decomplexed, is, according to this theory, thus reattached onlyto a subsidiary extent to the silver-loaded cation exchanger and themajority remains in the supernatant. The purity of the product fattyacid on the cation exchanger is considerably increased as a result.

[0070] According to the invention, therefore, heat can be supplied tothe system after complexing the fatty acid to the 100% silver-loadedcation exchanger, in order to ensure greater purity of the fatty acid.Preferably, this step is carried out at a temperature T>40° C. and apressure P=1 bar. Particularly preferably, the step is carried outbetween 40° C. and 80° C. Very particularly preferably between 50 and70° C., and very particularly preferably at from 55° C. to 65° C.

[0071] If temperatures of >40° C. are employed, the duration of thecomplexation reaction can be incremented in 30-minute steps, quitegenerally longer reaction times giving a higher product purity. However,it has been found that 2.5 h should be sufficient in most cases.

[0072] The use of acetonitrile as decomplexing agent can be omittedcompletely in the inventive method in the case of PUFA or PUFAderivatives having fewer than 4 double bonds, since conditions have beendeveloped in which

[0073] (i) the detachment succeeds in physiologically safe solvents and

[0074] (ii) a completely novel regeneration method has been developedfor the cation exchanger or the silver itself.

[0075] A solvent which can be used according to the invention for theattachment of the PUFA or of the derivative can be selected from thefollowing groups: alkanes, ketones, ethers, esters, diketones, diesters,diethers, diols, polyols, nitrites, dinitriles and alcohols;particularly suitable ketones are acetone and 2-methyl ethyl ketone[sic], particularly suitable alcohols are the medium-chain alcohols2-propanol, propanol, butanol, hexanol or isomers thereof, still moresuitable are the alkanes n-hexane, n-heptane, n-octane or isomersthereof, and most suitable is the nitrile acetonitrile, and also theshort-chain alcohols ethanol and methanol. Ethanol and methanol are thepreferred solvents.

[0076] According to the invention, the silver can, after each step ofattaching fatty acids to the ion exchanger, be detached by adding anacid (e.g HNO₃) or base (e.g. NaHCO₃). The complexed fatty acid is thusreleased at the same time. The silver precipitates out as salt (e.g.Ag₂CO₃) or remains in solution (AgNO₃) and can be separated off from theproduct fatty acid by decanting or extraction.

[0077] The silver can then be brought back into solution according tothe invention by adding a base (in the case of a preceding detachment byHNO₃, for example by adding NaHCO₃) or an acid (in the case of apreceding detachment by NaHCO₃, for example by adding HNO₃).

[0078] The following bases, inter alia, can be used according to theinvention: hydroxides and carbonates, organic bases, in particularsodium hydroxide, potassium hydroxide, preferably triethylamine, calciumcarbonate, magnesium carbonate, very particularly preferably potassiumcarbonate, potassium hydrogen carbonate, sodium carbonate and sodiumhydrogen carbonate.

[0079] The following acids, inter alia, can be used according to, theinvention: phosphoric acid, in particular hydrochloric acid, formic acidand sulfuric acid, very particularly preferably acetic acid and nitricacid.

[0080] As a result of the higher affinity of most ion-exchangermaterials toward Ag⁺ compared with other cations (Ag⁺>>Na⁺>>H⁺), thesilver is attached quantitatively to the cation exchanger. After variouswashing steps, the cation exchanger is reusable. The reattachment of thesilver is then at least 90%, preferably at least 95%, and veryparticularly preferably >99%, compared with the first attachment.

[0081] Using the inventive method, purification factors of from 1.01 to99 can be achieved, in which case, although these purification factorsdo depend on the method, they also depend on the concentration of thetarget compound in the starting mixture. Although therefore highpurification factors are expedient, it can nevertheless be of greatimportance to bring, for example, a target fatty acid from 80% purity to90%, precisely when, as shown in the examples, molecules are separatedfrom one another which only differ from one another in the presence of asingle further double bond. Although the corresponding purificationfactor in this example is “only” 1.125, in conjunction with the highinventive yields, this means a dramatic further development of allmethods and processes described in the prior art, for examplechromatographic methods and processes.

[0082] In the inventive method, in the case of PUFAs or PUFA derivativescontaining 4 or more double bonds, a fully silvered cation exchanger canbe used, from which the PUFAs are successfully detached usingacetonitrile, or else by regeneration of the cation exchanger or of theAg⁺, as described previously.

[0083] However, according to the invention, for PUFAs or PUFAderivatives having 4 or more double bonds, preferably a partiallysilvered cation exchanger can also be used, the detachment in this casebeing able to be performed by physiologically harmless solvents and/orheat, without the use of acetonitrile.

[0084] A solvent which can be used according to the invention fordetaching the PUFA or the derivative can be selected from the followinggroups: ketones (for example acetone, 2-methyl ethyl ketone [sic]),preferably medium-chain alcohols (for example 2-propanol, butanol,propanol), very particularly preferably short-chain alcohols (forexample methanol or ethanol) or mixtures of these solvents.

[0085] The invention relates to a method for fractionating a liquidmixture according to the degree of saturation of the compounds presentin the mixture. The degree of saturation depends on the number of doublebonds. The mixture can contain polyunsaturated organic compounds,unsaturated organic compounds, or else saturated organic compounds andalso inorganic compounds.

THE FIGURES BELOW EXPLAIN THE INVENTION IN MORE DETAIL:

[0086]FIG. 1 shows the principle of purifying PUFAs by selectiveextraction from the Ag⁺ ion exchanger according to the solvent which canbe used

[0087]FIG. 2 shows a plan of the inventive complexation of fatty acidethyl esters on the Ag⁺ ion exchanger

[0088]FIG. 3 shows a plan of the inventive regeneration of the silver

ABBREVIATIONS USED:

[0089] DHA All cis-4,7,10,13,16,19-docosahexaenoic acid

[0090] DPA All cis-4,7,10,13,16-docosapentaenoic acid (ω-6)

[0091] DHAEE All cis-4,7,10,13,16,19-docosahexaenoic acid ethyl ester

[0092] DPAEE All cis-4,7,10,13,16-docosapentaenoic acid ethyl ester(ω-6)

[0093] SFC Supercritical fluid chromatography

[0094] PUFA Polyunsaturated fatty acid

[0095] Terms Used:

[0096] Loading Capacity

[0097] The loading capacity of the cation exchanger describes the amountof PUFA or defined derivative (for example docosahexaenoic acid ethylester; DHAEE) in g or percent (%, w/w) which can be complexed to 100 gof the cation exchanger in the protonated form (H⁺ form).

[0098] The invention will be illustrated with reference to the followingexamples, without these being able to be considered as a limitation ofthe invention.

[0099] Unless stated otherwise, in all of the following examples thesubstrate used for the purification was a mixture of about 80% DHAEE and20% DPAEE (ω-6), which can be obtained from a microbial fermentationusing the strain Schizochytrium SR21 [Yokochi et al., Optimization ofdocosahexaenoic acid production by Schizochytrium limacinum SR21″, Appl.Microbiol. Biotechnol. (1998), 49: 72-76]. The Schizochytrium oilobtained from this fermentation was transesterified into the ethylesters and subjected to precipitation with urea, the saturated fattyacids being removed. DHAEE and DPAEE can be further purified by means ofHPLC [Yamamura, supra], and used as substrate for the purificationexperiments. Separation of the ethyl esters from one another inquantitatively significant amounts is a great problem, since the twofatty acids differ only in the differing number of double bonds.

EXAMPLE 1 Production of Amberlyst®15 (20-50 Mesh) 100% Loaded withSilver Ions

[0100] The silver-loaded ion exchangers are produced on the basis of themethod described by Nieto et al. (Nieto, S.; A. M. Cordoba; J. Sanhuenzyand A. Valenzuela (1997): Obtention of highly purified fractions ofeicosapentaenoic acid and docosahexaenoic acid from sardine oil bysilver-resin chromatography: A semipreparative procedure. Grasas yAceites, 48(4), 197-199) for Dowex® 50WX8 (earlier name Dowex®W-HCR-W2). However, the method for this was considerably simplified andmodified.

[0101] Neither is the material prepared in a heatable glass column, noris the material prewashed with organic solvents. However, in theinventive novel method, in contrast to the method disclosed by Nieto etal., the particle size of the material is be [sic] decisive for thequality of the separation and yield.

[0102] 20 g of Amberlyst® 15 are placed in a vacuum filter or glasscolumn equipped with a vacuum filter and washed with 1 M sodium nitratesolution (NaNO₃) until the pH of the eluate turns from acidic toneutral. Neutralization indicates reduced formation of nitric acid whichis formed on exchange of protons to release sodium ions. If the cationexchanger is completely loaded with sodium ions, the eluent remainsneutral.

[0103] Two different procedures can then be followed.

[0104] Either the sodium-loaded cation exchanger is washed with 0.4 Msilver nitrate solution until silver can be detected in the eluent, orthe material is first transferred by rinsing with sodium nitratesolution into a round-bottomed flask or conical flask. The excess sodiumnitrate solution is then discarded.

[0105] Subsequently, 5.4 ml of 0.4 M silver nitrate solution/g ofAmberlyst® 15 are stirred for 8-12 h. The supernatant is removed.

[0106] In both cases the procedure with the Ag⁺-loaded cation exchangeris as follows:

[0107] The silver-loaded cation exchanger (approximately 2.0 mmol ofAg⁺/ml of H⁺ exchanger, where 1 g of H⁺ Dowex is equivalent toapproximately 0.9 ml of H⁺ Dowex) is washed to be silver free, threetimes with 100 ml of water, and then washed to be water free twice with100 ml of ethanol (1 h). It is then left overnight (12 h) in 100 ml ofacetonitrile. The material is then washed again twice, each time with100 ml of ethanol. The material can then be used. Acetonitrile can alsobe replaced by using 100 ml of ethanol three times.

[0108] A different washing procedure or activation procedure can lead toless active or nonactive silver-loaded cation exchanger. The watercontent in the silver-loaded cation exchanger is of particularimportance here.

EXAMPLE 2 Production of Amberlyst® 15 (20-50 Mesh) Ion Exchanger whichis Partially Loaded, to 50%, with Silver Ions

[0109] Partially silvered Amberlyst® material is prepared with stirringin order to enable a uniform distribution of the silver on the cationexchanger.

[0110] 50% silvered Amberlyst® 15 material is prepared by adding 2.7 mlof 0.4 M silver nitrate solution/g of Amberlyst® 15 for 8-12 h. Thebatch will be stirred for 12 h. The supernatant is then decanted and thesilver-loaded cation exchanger is washed three times usingtwice-distilled water. The material is then washed twice with 100 ml ofethanol (1 h each time) and washed once with 100 ml of acetonitrile (12h). The acetonitrile can be replaced by multiple use of ethanol. Thematerial is finally taken up in ethanol and then used.

[0111] In the same way, material of 0.1-99.9% (based on 100% silvering)silvering can be prepared.

Various Detachment Protocols (FIG. 1) Variant A. Detaching the Productsfrom the Ag⁺ Exchanger Using a π-Electron Donor

[0112] The solvent which can detach the product ester from thesilver-loaded cation exchanger must be chosen depending on the exchangermaterial, silver loading and the attached fatty acid ethyl ester (numberof double bonds). In the ideal case, a solvent can be used whichlikewise acts as π-electron donor. Examples of this are acetonitrile,1-hexene or toluene.

[0113] For example, bound DHAEE can be separated off from thesilver-loaded cation exchanger in the most simple manner by addingacetonitrile (ACN). The acetonitrile in this case is a strongerπ-electron donor than the PUFAs.

[0114] For this, 100 ml of ACN are added to the batch and the mixture isstirred at room temperature under a protective gas. After completedetachment of the fatty acid ethyl ester, the organic phase is takenoff. The exchanger material is washed again with ACN and the organicphases are combined. After taking off the solvent, the purified DHAEE isobtained as a slightly yellow oil. In all of the examples listed, justrelatively small amounts of acetonitrile are sufficient for this, forexample a mixture of acetonitrile in n-hexane (10:90, v:v).

[0115] Examples 3-9 below are examples of such methods in which ACN isused to detach the complexed fatty acid from the exchanger.

EXAMPLE 3 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Amberlyst® 15 (20-50 Mesh) in Ethanol over 24 Hours:

[0116] In preliminary experiments it was found that the PUFA loadingcapacity of the exchanger is approximately 5% by weight of PUFA (g ofPUFA/100 g of H⁺ exchanger).

[0117] 597.8 mg of docosahexaenoic acid ethyl ester (DHAEE) and 144.0 mgof docosapentaenoic acid ethyl ester (ω-6 DPAEE) were added to a stirredsolution of the 100% silver-loaded exchanger (10 g of H⁺ Amberlyst® 15)from example 1 in 100 ml of absolute ethanol. The suspension is shakenat 100 rpm for 24 h at room temperature under a protective gas. Anantioxidant may be further added (e.g. tocopherols,2,6-di-tert-butyl-4-methylphenol (BHT), ascorbyl palmitate) to avoidoxidation of the PUFAs.

[0118] After time intervals of 30 or 60 min in each case, the reactionis assessed quantitatively and qualitatively by analyzing thesupernatant by gas chromatography (HPGC6890, column: Macherey & NagelFFAP Permabond 0.1 μm (25 m, 0.25 mm), with splitting (10:1), carriergas: helium (constant flow 1.0 ml/min), FID operation using hydrogen (30ml/min) and oxygen (300 ml/min) as fuel gases, make up: 20 ml of helium,detector and injector temperatures: in each case 255° C., GC furnacetemperature program: initial temperature 180° C., temperature rise rate10° C./min to a final temperature of 230° C., hold this for 5 min,injection volume: 1.0 μl). By adding to the reaction batch an internalstandard which does not bind to the silver-loaded cation exchanger (forexample a saturated fatty acid or an ester of a saturated fatty acid, oranother derivative), quantitative analysis of the polyunsaturated fattyacids in the supernatant can be carried out.

[0119] The theoretical loading of the exchanger can be calculated inthis case by subtracting the fatty acid ethyl ester remaining in thesupernatant from the total fatty acid ethyl ester used. The purity ofthe product can likewise be determined in this way (table 1).

[0120] In this case, virtually all of the DHAEE (86.7%) was complexed onthe exchanger. The purity of the DHAEE was increased from 80% tovirtually 91%. The complexation is complete after only a few hours.

[0121] When the ion exchanger is completely loaded with PUFAs (furtherattachment no longer observable) or the desired purity (e.g. DHAEE >90%)is achieved, the supernatant is taken off, and the ion exchangermaterial is washed once with 100 ml of ethanol and freed from residuesof unbound fatty acid ethyl ester. After the reaction is complete, thebound product fatty acid ethyl ester can be detached from thesilver-loaded cation exchanger by various methods.

[0122] Most simply, DHAEE can be detached by stirring with 100 ml ofacetonitrile for 7 h under a protective gas. For this, simply a 10%strength mixture of acetonitrile in ethanol or n-hexane is sufficient.After removing the solvents, 482.6 mg of the purified DHAEE wereisolated at a purity of 88.4%.

[0123] This value agrees well with the calculated value from table 1.The yield of DHAEE is 81%. The product loading of the exchanger is thus4.8% (g of DHAEE/100 g of H⁺ exchanger).

[0124] From the supernatant, after removing the solvents, a further 66.4mg of DHAEE were isolated. The overall DHAEE recovery was 92%. TABLE 1Results of attaching a mixture of DHAEE and DPAEE to Ag⁺ Amberlyst ® 15DHAEE S S S S R in DHAEE DPAEE DHAEE yield Purification Time DPAEE DHAEEDPAEE DHAEE Total EtOH Amberlyst Amberlyst Amberlyst Amberlyst factor(h) (area %) (area %) (mg) (mg) (mg) (%) (mg) (mg) (area %) (%) DHAEE 020.5 79.5 146.6 567.7 714.3 96.3 30.1 −2.5 109.2 5.0 1 39.0 61.0 100.7158.3 259.0 34.9 439.4 43.3 91.0 73.5 1.14 2 45.0 55.0 99.0 121.4 220.429.7 476.4 45.0 91.4 79.7 1.14 3 46.4 53.6 100.8 116.6 217.4 29.3 481.243.3 91.8 80.5 1.15 7 50.8 49.2 96.8 94.0 190.8 25.7 503.8 47.2 91.484.3 1.14 24 53.6 46.4 91.8 79.4 171.2 23.1 518.4 52.2 90.8 86.7 1.14

EXAMPLE 4 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Amberlyst® 15 (20-50 Mesh) in Ethanol over 210 Minutes

[0125] For this, a mixture of 0.585 g of docosahexaenoic acid ethylester (DHAEE) and 0.146 g of docosapentaenoic acid ethyl ester (ω-6DPAEE) is added to a stirred solution of the 100% silver-coatedexchanger (equivalent to 10 g of H⁺ Amberlyst® 15) in 100 ml of absoluteethanol. The suspension is shaken at 100 rpm for 210 min at 26° C. undera protective gas. At the times indicated, the supernatant was analyzedby gas chromatography.

[0126] The purity and yield of the product can likewise be determined inthis manner (table 2).

[0127] Compared with the experimental attachment using Dowex® 50WX8200-400 mesh (example 6, table 4), a considerably reduced reaction timeand a marked increase of the DHAEE loading from 1.2% (g of DHAEE/100 gof H⁺ Dowex 50WX8) to 4.6% (g of DHAEE/100 g of H⁺ Amberlyst®) wasobserved. The purity of the DHAEE was increased from 80% to over 92.9%.The yield of 92.9% pure DHAEE is 77.9%. The comparable exchanger(particle size) from the Dowex® 50WX8 series (20-40 mesh) does not showany DHAEE loading capacity and, therefore, also no purifying effect.TABLE 2 Results of attaching a mixture of DHAEE and DPAEE to Ag⁺Amberlyst ® 15 (20-50 mesh) S S S S R in DHAEE DPAEE DHAEE DHAEEPurification Time DPAEE DHAEE DPAEE DHAEE Total EtOH Amberlyst AmberlystAmberlyst yield factor (min) (area %) (area %) (mg) (mg) (mg) (%) (mg)(mg) (area %) (%) DHAEE 0 20.6 79.4 180.9 696.4 877.3 120.0 −111.4 −34.976.1 −19.0 10 25.6 74.4 130.7 380.2 510.9 69.9 205.0 15.3 93.0 35.0 1.1620 29.1 70.9 119.2 290.3 409.5 56.0 294.8 26.8 91.7 50.4 1.15 30 32.367.7 118.8 248.8 367.5 50.3 336.3 27.2 92.5 57.5 1.16 45 35.5 64.5 117.1212.2 329.3 45.0 372.9 28.9 92.8 63.7 1.16 60 37.3 61.2 117.3 192.5309.7 42.4 392.7 28.7 93.2 67.1 1.17 150 44.2 55.8 114.4 144.5 258.935.4 440.6 31.6 93.3 75.3 1.17 210 43.9 51.0 111.4 129.5 240.8 32.9455.7 34.7 92.9 77.9 1.16

EXAMPLE 5 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Amberlyst® 15 (20-50 Mesh) in n-hexane:

[0128] For this, 585.1 mg of docosahexaenoic acid ethyl ester (DHAEE)and 146.0 mg of docosapentaenoic acid ethyl ester (ω-6 DPAEE) are addedto a stirred solution of the completely silver-loaded exchanger (9.3 gof H⁺ Amberlyst® 15) in 100 ml of n-hexane. The suspension is shaken at100 rpm for 3.5 h at room temperature under a protective gas.

[0129] After time intervals of 30 or 60 min in each case, the reactionis assessed quantitatively and qualitatively by analyzing thesupernatant by gas chromatography. The theoretical loading of theexchanger can be calculated here by subtraction. The purity of theproduct can likewise be determined in such a manner (table 3).

[0130] The purity of the DHAEE was increased 80% to over 94.5%. Theaddition is virtually complete after only a few minutes. The loading ofthe exchanger with DHAEE is 4.4% of DHAEE/100 g of H⁺ exchanger). Theyield of 94.5% pure DHAEE is 74.6%.

[0131] The supernatant is taken off, the cation exchanger material iswashed once with 100 ml of n-hexane and freed from residues of unboundfatty acid ethyl ester. After the reaction is complete, the boundproduct fatty acid ethyl ester can be detached from the silver-loadedcation exchanger by various methods.

[0132] Most simply, DHAEE can be detached by stirring with 100 ml ofacetonitrile for 7 h under a protective gas. Instead of the pureacetonitrile, however, just a 10% strength mixture of acetonitrile inn-hexane can also be used, which achieves an identical result. TABLE 3Results of attaching a mixture of DHAEE and DPAEE to Ag⁺ Amberlyst ® 15S S S S R in DHAEE DPAEE DHAEE DHAEE Purification Time DPAEE DHAEE DPAEEDHAEE Total EtOH Amberlyst Amberlyst Amberlyst yield factor (min) (area%) (area %) (mg) (mg) (mg) (%) (mg) (mg) (area %) (%) DHAEE 0 20.7 79.3158.8 609.1 767.9 105.0 −24.0 −12.7 65.3 −4.1 10 29.0 71.0 118.2 290.1408.3 55.8 295.1 27.9 91.4 50.4 1.14 20 32.9 67.1 127.5 260.5 388.0 53.1324.6 18.5 94.6 55.5 1.18 45 37.6 62.4 121.7 202.3 324 44.3 382.9 24.394.0 65.4 1.18 60 39.1 60.9 123.4 192.5 315.9 43.2 392.7 22.6 94.6 67.11.18 150 43.2 56.8 121.9 160.4 282.3 38.6 424.8 24.1 94.6 72.6 1.18 21044.9 55.1 120.7 148.4 269.1 36.8 436.8 25.3 94.5 74.6 1.18

EXAMPLE 6 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Amberlyst® 15 in n-hexane at Elevated Temperature:

[0133] For this, 1.18 g of docosahexaenoic acid ethyl ester (DHAEE) and0.29 g of docosapentaenoic acid ethyl ester (ω-6 DPAEE) are added to astirred solution of the completely silver-loaded exchanger (9.3 g of H⁺Amberlyst®) in 100 ml of n-hexane. The suspension is shaken at 100 rpmat room temperature [sic] under a protective gas for 2 h at 55° C. Ifappropriate, an antioxidant can further be added (e.g. tocopherol,ascorbyl palmitate), to prevent oxidation of the PUFAs.

[0134] After 30 or 60 min time intervals in each case, the reaction isassessed quantitatively and qualitatively by analyzing the supernatantby gas chromatography. The theoretical loading of the exchanger can becalculated by subtraction here. The purity of the product can likewisebe determined in this manner (table 4).

[0135] The purity of the DHAEE was increased from 79% to 97.9%. Theattachment is complete after only a few minutes. The loading of theexchanger with DHAEE is 5.5% (g of DHAEE/100 g of H⁺ exchanger). TheDHAEE yield is 47.1%. TABLE 4 Results of attaching a mixture of DHAEEand DPAEE to Ag⁺ Amberlyst ® 15 at 55° C. S S S S R in DHAEE DPAEE DHAEEDHAEE Purification Time DPAEE DHAEE DPAEE DHAEE Total n-hexane AmberlystAmberlyst Amberlyst yield factor (min) (area %) (area %) (mg) (mg) (mg)(%) (mg) (mg) (area %) (%) DHAEE 0 20.5 78.9 312.7 1205.6 1518.3 103.0−25.9 −18.4 58.6 −2.2 10 25.8 73.5 300.2 855.7 1155.9 78.4 324.1 −5.8101.8 27.5 1.27 20 27.1 72.9 288.0 775.2 1063.2 72.1 404.5 6.4 98.4 34.31.23 30 27.8 72.2 283.5 736.7 1020.2 69.2 443.0 10.9 97.6 37.6 1.22 4528.6 71.4 292.9 731.6 1024.4 69.5 448.2 1.5 99.7 38.0 1.25 60 29.5 70.5290.5 693.7 984.2 66.8 486.0 3.9 99.2 41.2 1.24 120 30.9 68.3 282.6623.9 906.5 61.5 555.8 11.8 97.9 47.1 1.22

EXAMPLE 7 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Dowex® 50WX8 (200-400) Mesh [sic]:

[0136] A study was also conducted to establish what influence the meshsize of the exchanger material used has on the separation.

[0137] For this, 291.4 mg of docosahexaenoic acid ethyl ester (DHAEE)and 72.7 mg of docosapentaenoic acid ethyl ester (ω-6 DPAEE) are addedto a stirred solution of the completely silver-loaded exchanger (21 g ofH⁺ Dowex® 50WX8, 200-400 mesh) in 100 ml of absolute ethanol. Thesuspension is stirred at room temperature under a protective gas for 24h at 300 rpm using a magnetic stirrer.

[0138] After 30 or 60 min time intervals in each case, the reaction isassessed quantitatively and qualitatively by analyzing the supernatantby gas chromatography. The theoretical loading of the exchanger can becalculated by subtraction here. The purity of the product can likewisebe determined in such a manner (table 5).

[0139] The purity of the DHAEE was increased from 80% to 91.7%. Theattachment is complete after 46 hours. The loading of the exchanger withDHAEE is 1.2% (g of DHAEE/100 g of H⁺ exchanger). The yield of the 91.7%pure DHAEE is 83.7%.

[0140] In contrast to this, the DHAEE loadings with the fully silveredDowex® 50WX8 100-200 mesh material are 1.1% (g of DHAEE/100 g of H⁺exchanger) and with the fully silvered Dowex® 50WX8 20-40 mesh materialare virtually 0% DHAEE/H⁺ exchanger. Here, therefore, a pronounceddependence on the particle size of the exchanger material is found.TABLE 5 Results of attaching a mixture of DHAEE and DPAEE to Ag⁺ Dowex ®50 WX8 (200-400 mesh) S S S S R in DHAEE DPAEE DHAEE DHAEE PurificationTime DPAEE DHAEE DPAEE DHAEE Total EtOH Dowex Dowex Dowex yield factor(h) (area %) (area %) (mg) (mg) (mg) (%) (mg) (mg) (area %) (%) DHAEE 017.7 73.2 66.9 277.6 344.5 94.6 13.8 5.7 70.6 4.8 1 19.6 68.8 65.9 229.0294.8 81.0 62.4 6.8 90.1 21.4 1.13 2 21.1 68.5 66.9 216.0 283.0 77.775.4 5.7 92.9 25.9 1.16 3 21.7 66.0 65.9 199.8 265.7 73.0 91.6 6.8 93.131.4 1.16 5 23.3 63.7 63.7 175.0 238.7 65.5 116.4 9.0 92.8 40.0 1.166.25 24.2 62.3 63.7 163.1 226.8 62.3 128.3 9.0 93.4 44.0 1.17 7 24.761.6 63.7 157.7 221.4 60.8 133.7 9.0 93.7 45.9 1.17 22 31.7 50.4 57.291.9 149.0 40.9 199.6 15.4 92.8 68.5 1.16 28 34.7 46.8 55.1 74.5 129.635.6 216.9 17.5 92.5 74.4 1.16 46 41.1 38.7 50.8 47.5 98.3 27.0 243.921.9 91.7 83.7 1.15

EXAMPLE 8 Experimental Attachment of a Mixture of DHAEE and DPAEE to Ag⁺Dowex® 50WX8 with Subsequent Concentration by Temperature Shift:

[0141] For this, 116.7 mg of docosahexaenoic acid ethyl ester (DHAEE)and 29.2 mg of docosapentaenoic acid ethyl ester (ω-6 DPAEE) are addedto a stirred solution of the completely silver-loaded exchanger (25 g ofH⁺ Dowex® 50WX8, 100-200 mesh) in 200 ml of absolute ethanol. Thesuspension is stirred at room temperature under a protective gas for -24h at 300 rpm using a magnetic stirrer. If appropriate, an antioxidantcan further be added (e.g. tocopherol, ascorbyl palmitate), to preventoxidation of the PUFAs.

[0142] After 30 or 60 min time intervals in each case, the reaction isassessed quantitatively and qualitatively by analyzing the supernatantby gas chromatography. The theoretical loading of the exchanger can becalculated here by subtraction. The purity of the product can likewisebe determined in such a manner (table 6).

[0143] The purity of the DHAEE was increased from 80% to more than92.8%. The attachment is complete after 24 hours. The loading of theexchanger with DHAEE is 0.40% (g of DHAEE/100 g of ⁺H exchanger).

[0144] A further increase in the purity of the DHAEE on thesilver-loaded cation exchanger is obtained by washing the PUFA-loadedcation exchanger with 100 ml of solvent (for example ethanol) and thenheating it in a solvent (for example ethanol, acetone). The temperaturerange used here is between 50° C. and 80° C. (table 7). TABLE 6 Resultsof attaching a mixture of DHAEE and DPAEE to Ag⁺ Dowex ® 50 WX8 S S S SR in DHAEE DPAEE DHAEE DHAEE Purification Time DPAEE DHAEE DPAEE DHAEETotal EtOH Dowex Dowex Dowex yield factor (h) (area %) (area %) (mg)(mg) (mg) (%) (mg) (mg) (area %) (%) DHAEE 0 19.3 77.8 30.2 118.8 149.0101.5 −2.1 −0.2 89.6 −1.7 1 21.8 74.0 28.1 95 123.1 84.2 21.7 1.3 94.518.6 1.18 2 24.1 72.4 28.1 82.1 101.2 74.9 34.7 1.9 94.7 29.7 1.18 3.526.1 70.0 25.9 71.3 97.2 66.6 45.5 3.2 93.4 39.0 1.17 4.5 27.3 68.6 25.964.8 90.7 62.0 55.0 3.5 93.8 44.5 1.17 5.5 27.5 64.3 25.9 60.5 86.4 59.256.3 3.2 94.6 48.2 1.18 6.5 30.0 65.2 25.9 54.0 79.9 54.0 62.8 4.3 93.653.8 1.17 7.5 30.3 61.2 23.8 49.7 73.5 50.3 67.1 5.4 92.6 57.5 1.16 2447.2 37.9 21.6 17.3 38.9 26.5 99.5 7.8 92.8 85.2 1.16

[0145] TABLE 7 Development of the purities of DHAEE and DPAEE in thesupernatant (ethanol) during heating to differing temperatures. 60° C.70° C. 80° C. t DPAEE DHAEE DPAEE DHAEE DPAEE DHAEE [h] [area %] [area%] [area %] [area %] [area %] [area %] 0.5 15.3 84.7 12.5 87.5 13.4 86.61 18.0 82.0 15.3 84.7 15.9 84.1 1.5 21.1 78.9 17.2 82.8 18.4 81.6 2 21.978.1 19.1 80.9 19.1 80.9 2.5 25.0 75.0 22.3 77.7 22.6 77.4 Product 5.095.0 4.6 95.4 4.9 95.3 phase ACN Starting 7.7 92.3 7.7 92.3 7.7 92.3material on Dowex 50 WX8

[0146] For this, a Pyrex tube (Pyrex borosilicate glass tubes 16×100 mm,Merck Eurolab, Darmstadt) is charged each time with 1 ml of Ag-Dowex® 50WX8 (200-400 mesh), which has been loaded with 1.21% DHAEE (g ofDHAEE/100 g of H⁺ Dowex), and mixed with 2 ml of ethanol and mixed onthe Vibromixer. The tube is then heated to 60, 70 and 80° C.) [sic] andthe supernatant is analyzed by gas chromatography. The purity of theDHAEE on the Dowex at the start of the experiment was 92.3% (Dowex® 50WX8, 200-400 mesh). After 2.5 h, it is allowed to cool, the supernatantis taken off and washed once with 2 ml of ethanol. The DHAEE is thendecomplexed from the Dowex using 2 ml of ACN for 3 h.

[0147] The gas-chromatographic analysis of the ACN phase shows anenrichment of the DHAEE on the silver-loaded cation exchanger toapproximately 95% in all experiments.

[0148] Since detachment of the product by acetonitrile is undesirable inmany cases, other methods were developed in which the use of a toxicsolvent can be avoided, or the use can be ruled out completely. Thesemethods are explained below.

Variant C Detaching the Products from the Ag⁺ Exchanger without ACN(FIG. 1)

[0149] In the case of complexing with PUFAs having <4 double bonds, thePUFAs can be successfully detached from the 100% silver-loaded cationexchanger just by adding a polar solvent (for example ethanol in thecase of attachments in nonpolar solvents such as n-hexane) without orwith simultaneous temperature increase (for example heat exchanger,heating bath, microwave, thermal block), without using ACN.

[0150] Generally, the temperatures are just below the boiling point ofthe solvent used. Should the PUFA be too strongly complexed, a cationexchanger can be used which is not 100% loaded with silver and thereforeis unable to complex the PUFAs so strongly. In many cases, separatingoff the PUFAs succeeds just by using one of the abovementioned solventswithout temperature shift.

EXAMPLE 9 Experimental Attachment of a Mixture of DHAEE and DPAEE to 30%Partially Silvered Ag⁺ Amberlyst® 15 (20-50 Mesh)

[0151] For this, a mixture of 0.63 g of docosahexaenoic acid ethyl ester(DHAEE) and 0.17 g of docosapentaenoic acid ethyl ester ((ω-6 DPAEE) isadded to a stirred solution of the 30% silver-loaded exchanger (10 g ofH⁺ Amberlyst® 15) in 100 ml of n-hexane. The suspension is shaken at 26°C. under a protective gas for 60 min at 100 rpm.

[0152] [lacuna] The purity of the product can likewise be determined insuch a manner (table 8).

[0153] The purity of the DHAEE was increased from 80% to 90.5%. Theattachment is complete after just a few minutes. The DHAEE loading ofthe exchanger is 2.1% (g of DHAEE/100 g of H⁺ exchanger). Thetheoretical yield from the table is 32.5% of 90.5% pure DHAEE.

[0154] Detaching the product: after removing the hexane phase togetherwith the unbound product, 100 ml of ethanol are added to the exchanger,the mixture is stirred for 30 min at room temperature and the ethanolphase is taken off. From this phase, already 176.3 mg of DHAEE (purity87.6% area) may be isolated. The exchanger is then stirred with afurther 100 ml of ethanol at 70° C. for 1 h at 300 rpm. From this phase,a further 51.6 mg of DHAEE (94.5% area) can be isolated. Fractionationof the ethanol phases, however, is not necessary. From the separatedhexane phase, after removing the hexane, 379.9 mg of DHAEE werereisolated. The recovery of the bound DHAEE is greater than 96%. Thecombined ethanol extracts lead to 227.9 mg of DHAEE at a purity of 89.2%(area). The yield of DHAEE is 32%. TABLE 8 Results of attaching amixture of DHAEE and DPAEE to partially silvered Ag⁺ Amberlyst ® 15(20-50 mesh) S S S S R in DHAEE DPAEE DHAEE DHAEE Purification TimeDPAEE DHAEE DPAEE DHAEE Total EtOH Amberlyst Amberlyst Amberlyst yieldfactor (min) (area %) (area %) (mg) (mg) (mg) (%) (mg) (mg) (area %) (%)DHAEE 0 19.2 71.5 161.7 601.3 763.0 95.4 29.4 7.7 79.2 4.7 10 22.3 65.8141.4 416.9 558.1 69.8 213.8 28.1 88.4 33.9 1.11 30 22.9 66.0 151.1435.5 586.7 73.3 195.2 18.3 91.5 30.9 1.14 60 22.8 65.7 147.9 425.6573.5 71.7 205.1 21.5 90.5 32.5 1.13

Variant B Detaching the Silver from the Cation Exchanger withSimultaneous Release of the Fatty Acid Ethyl Ester (FIGS. 1 and 3)EXAMPLE 10 Regenerative Detachment of the Fatty Acid Ethyl Ester

[0155] To avoid the use of acetonitrile when detaching the DHAEE fromthe Ag⁺ Dowex®, the processes as described in FIG. 2 can be used.

[0156] After separating off the ethanolic supernatant from the reaction,the silver-loaded cation exchanger is washed silver-free with an excessof 0.4 M sodium nitrate solution (NaNO₃). This can be performed in around-bottomed flask (batch) or in a glass column (continuously). At thesame time, the DHAEE is released from the cation exchanger and can bedecanted off from the aqueous phase as an oily phase. It is notnecessary here to leach off all the silver to detach the DHAEE. However,in practice, complete detachment is the simplest method.

[0157] After separating off the product, the silver is precipitated outwith a carbonate solution (e.g. Na₂CO₃, K₂CO₃ or NaHCO₃). The silvercarbonate formed (e.g. Ag₂CO₃) precipitates out as a yellow precipitateand can be filtered off from the supernatant in a very simple manner.The silver carbonate is then brought into solution with stoichiometricamounts of nitric acid (HNO₃). This solution can be used directly forreattaching the silver.

[0158] The silver loading of the exchanger corresponds to the initialloading. The selectivity of the exchanger is retained in full.

[0159] In this manner, the detachment and isolation of the DHAEE can beperformed without solvent, which is of great advantage particularly forapplications in foods.

1. a method for separating off one or more of the most highlyunsaturated compound(s) from a liquid mixture additionally comprisingone or more less highly unsaturated organic compound(s) and/or one ormore saturated organic compound(s), characterized in that (i) an acidcation exchanger is loaded with silver ions, (ii) the loaded cationexchanger is mixed with the liquid mixture, (iii) in a batch method, themixture is stirred at a temperature which is below the boiling point ofthe solvent used, (iv) the supernatant is separated off, and (v) themost highly unsaturated compound(s) is (are) detached from the cationexchanger:
 2. The method as claimed in claim 1, characterized in thatthe most highly unsaturated compound is a polyunsaturated fatty acidhaving at least two double bonds.
 3. The method as claimed in claim 1 or2, characterized in that the most highly unsaturated compound(s) is(are) detached from the cation exchanger by adding a solvent selectedfrom the group consisting of ketones, alcohols and nitrites, or amixture of such solvents.
 4. The method as claimed in claim 3,characterized in that the most highly unsaturated compound(s) is (are)detached from the cation exchanger by adding acetonitrile, methanol orethanol.
 5. The method as claimed in claim 1 or 2, characterized in thatthe most highly unsaturated compound(s) is (are) detached from thecation exchanger by detaching the silver from the ion exchanger byadding sodium nitrate.
 6. The method as claimed in claim 3,characterized in that the most highly unsaturated compound(s) is (are)detached from the cation exchanger at a temperature range from −20° C.to 80° C.
 7. The method as claimed in one of the preceding claims,characterized in that the liquid mixture comprises a solvent.
 8. Themethod as claimed in claim 7, characterized in that the solvent isselected from the group consisting of ethers, esters, ketones, alkanes,alcohols and nitriles.
 9. The method as claimed in claim 7,characterized in that the solvent is selected from the group consistingof n-hexane, n-heptane, n-pentane, ethanol, methanol and acetonitrile.10. The method as claimed in one of the preceding claims, characterizedin that the liquid mixture comprises a saturated fatty acid and/or anunsaturated fatty acid containing n double bonds and/or a fatty acidcontaining n+1 or more double bonds, where the fatty acid containing n+1or more double bonds is separated from the saturated fatty acid or theunsaturated fatty acid containing n double bonds, where n is an integerbetween 1 and
 10. 11. The method as claimed in one of the precedingclaims, characterized in that the liquid mixture comprises crude estermixtures, processed oils, ester mixtures, fatty acid mixtures and/orderivatives of polyunsaturated fatty acids.
 12. The method as claimed inone of the preceding claims, characterized in that the liquid mixturecomprises fish oils, vegetable oils and/or microbial oils.
 13. Themethod as claimed in one of claims 2 to 12, characterized in that thepolyunsaturated fatty acid is selected from the group consisting ofhexadecadienoic acids, hexadecatrienoic acids, hexadecatetraenoic acids,linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid,arachidonic acid, eicosatrienoic acids, eicosatetraenoic acids,eicosapentaenoic acids, docosapentaenoic acids, docosahexaenoic acid,tetracosadienoic acids, octacosaoctaenoic acids.
 14. The method asclaimed in one of the preceding claims, characterized in that the acidcation exchanger is selected from the group consisting of Dowex® 50 WX8,Dowex® 50 WX4, Dowex® 50 WX2, Dowex® MWC1, Dowex® MSC1, Dowex®Monosphere C-350, Dowex® CCR-2, Dowex® DR 2030, Amberlite® CG50,Amberlite® IR-120, Amberlyst® 15, Bio-Rex® 70 Resin, Macherey & NagelPS-DVB®.
 15. The method as claimed in one of the preceding claims,characterized in that the acid cation exchanger is loaded by treatmentwith silver nitrate.
 16. The method as claimed in one of the precedingclaims, characterized in that the mixture of loaded cation exchangerwith the liquid mixture is stirred at elevated temperature.
 17. Themethod as claimed in claim 16, characterized in that the temperature iselevated by the action of an external heat source, by microwaves,ultrasound or electromagnetic radiation.
 18. The method as claimed inone of claims 1 to 17, characterized in that the liquid mixturecomprises at least one polyunsaturated fatty acid containing at least 4double bonds and the acid cation exchanger is completely loaded withsilver ions and the most highly unsaturated compound(s) is (are)detached from the cation exchanger by adding acetonitrile.
 19. Themethod as claimed in one of claims 1 to 17, characterized in that theliquid mixture comprises at least one polyunsaturated fatty acidcontaining at least 4 double bonds and the acid cation exchanger iscompletely loaded with silver ions and the most highly unsaturatedcompound(s) is (are) detached from the cation exchanger by detaching thesilver ions.
 20. The method as claimed in claim 19, characterized inthat the silver ions are detached from the cation exchanger by addingsodium nitrate.
 21. The method as claimed in one of claims 1 to 17,characterized in that the liquid mixture comprises at least onepolyunsaturated fatty acid containing at least 4 double bonds and theacid cation exchanger is only partially loaded with silver ions and themost highly unsaturated compound(s) is (are) detached from the cationexchanger by adding a solvent selected from the group consisting of theketones and alcohols.
 22. The method as claimed in one of claims 1 to17, characterized in that the liquid mixture comprises at least onepolyunsaturated fatty acid containing fewer than 4 double bonds and theacid cation exchanger is completely loaded with silver ions and the mosthighly unsaturated compound(s) is (are) detached from the cationexchanger by adding a solvent selected from the group consisting of theketones and alcohols.
 23. The method as claimed in claim 21 or 22,characterized in that the most highly unsaturated compound(s) is (are)detached from the cation exchanger by adding methanol or ethanol.